Opaganib与奥沙利铂和多柔比星联用作为复发/难治性高危神经母细胞瘤的新型挽救治疗

The combination of oxaliplatin and doxorubicin (OXDOX) has shown efficacy in heavily pre-treated neuroblastoma (NB) patients. Nevertheless, addition of agents that enhance response to OXDOX may improve outcomes. DOX has been shown to up-regulate the expression of the Sphingosine kinases (SphK1 and SphK2) and enhance the production of pro-survival sphingosine-1-phosphate (S1P) at the expense of pro-apoptotic ceramide (Cer) which, in turn, mediates resistance to DOX. Elsewhere, studies have shown that the SphKs regulate the sensitivity of cells to OX. Inhibition of SphK activity decreases S1P production and may reverse OXDOX resistance. The purpose of this study was to determine whether the addition of the SphK2 inhibitor, opaganib, could improve the efficacy of OXDOX salvage therapy for high risk relapsed/refractory neuroblastoma (HRNB) patients. An immunocompetent mouse model of HRNB was utilized with 9464-D M2 cells grown subcutaneously in syngeneic C57BL/6 mice, to evaluate the efficacy of the combined treatment relative to OXDOX and opaganib alone cohorts. Opaganib has been shown to reduce c-Myc and n-Myc expression. Western blot analysis evaluated the expression of n-Myc, Mcl-1, and markers of apoptosis including cleaved Caspase 3 and cleaved PARP in response to single agent and combination treatments. The reduction of MYCN in MYCN amplified NB patients was further explored to examine the ability of opaganib to directly target engage n-Myc using the Cellular Thermal Shift (CETSA) assay and Isothermal Dose-Response Fingerprint (ITDRF) assays. In the immunocompetent 9464-D mouse model, the combination of agents significantly reduced tumor size relative to opaganib alone and OXDOX alone cohorts (p<0.001). IHC staining demonstrated that the combination reduced proliferation (Ki-67), enhanced apoptosis (Caspase 3) and, enhanced macrophage infiltration (CD68) into the tumor microenvironment. Western blot analysis revealed that opaganib reduced n-Myc and Mcl-1 expression (> 5-fold). CETSA experiments confirmed direct target engagement of n-Myc by opaganib in live, whole cell assays. ITDRF assays determined that opaganib binds to n-Myc with micromolar affinity, consistent with its IC50 for cell viability. Time course assays demonstrated that opaganib rapidly bound n-Myc within 30 minutes of exposure. Together, these studies indicate that opaganib enhances the therapeutic efficacy of the OXDOX combination by both altering sphingolipid metabolic profiles to enhance the production of pro-apoptotic ceramide and by directly binding to and destabilizing n-Myc, a key oncogenic driver of high-risk NB. Opaganib has been extensively tested in adult COVID patient populations with a favorable toxicity profile. Clinical testing of this novel three-drug combination in NB patients is under evaluation by the Beat Childhood Cancer Research Consortium.